Proteomics refers to the qualitative and quantitative scientific analyses of the expression, localizations, functions and interactions of the proteins and peptides expressed by the genetic material of an organism. Proteomics and the technology commonly used therein find equal application in veterinary research, with other organisms and even non-living matter that contain appreciable quantities of proteins and/or peptides (e.g., soil, air). In proteomic research, proteins and peptides found in the human body, in other living organisms, and in non-living systems are analyzed for a wide array of purposes. For instance, in applications where proteins/peptides are collected from human subjects, proteomic analysis can be of great value in gaining a better understanding of metabolic pathways, in predicting the clinical outcome of therapeutic interventions to disease, or in elucidating the function of various organic molecules, to name but a few. There is significant potential with this discipline to expose heretofore unknown pathways and biochemical interactions that may lead to important findings in terms of human health and medicine. Indeed, many important biomarkers have been identified by application of this technology.
As noted above, one area of proteomic research involves the analysis of human proteins and peptides. With respect to mammalian subjects, and humans in particular, proteins and peptides may be readily collected through any number of methodologies and from any number of sources. By way of example, proteins and peptides may be found in mammalian body fluids, sera such as blood (including whole blood as well as its plasma and serum), CSF (spinal fluid), urine, sweat, saliva, tears, pulmonary secretions, breast aspirate, prostate fluid, seminal fluid, stool, cervical scraping, cysts, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumor tissue, hair, skin, buccal scrapings, nails, bone marrow, cartilage, prions, bone powder, ear wax, etc. or even from external or archived sources such as tumor samples (i.e., fresh, frozen or paraffin-embedded). Samples, such as body fluids or sera, obtained during the course of clinical trials may be particularly advantageous for use in connection with proteomic research, although samples obtained directly from living subjects under alternate conditions or for other purposes may be readily used as well.
Moreover, analysis of blood chemistry is routinely used in medicine for both diagnostic and prescriptive purposes. Currently, to analyze blood chemistry, a practitioner must order a series of tests or “panels” in which the concentration of a specific species or family of species within the blood serum is measured. For example, an electrolyte panel measures the concentrations of sodium, potassium, chloride, calcium and phosphorous. Performing such tests, however, can be time consuming and expensive. Furthermore, each panel focuses on a single species or family of species of interest, and is therefore limited in scope. It is therefore generally necessary for a practitioner to have, prior to ordering a panel, a suspicion of what species merit further analysis. In this sense, the practitioner must know ahead of time what he is looking for. A single test that returned a definitive blood chemistry signature upon which a wide range of diagnoses could be made and treatments prescribed would be very useful.
One approach to obtaining such a signature involves the direct determination of the concentration of all (or substantially all) species within the blood through mass spectroscopy. Difficulties arise, however, due to the extremely wide range of concentrations of species within the blood serum. In fact, regardless of the collection technique or the source of the proteins and peptides studied in connection with proteomics research, or the test used to collect a sample from a patient in obtaining a “panel,” one limitation of this type of analysis, in general, is the difficulty in discerning small variations in the levels of proteins and peptides of relatively low abundance in a sample. For instance, there are thousands of different proteins and peptides present in a sample of serum obtained from a human subject. Some of these proteins and peptides are present in relatively high abundance, while others are present in relatively low abundance. Certain proteins of high abundance, such as albumin, immunoglobulin and transferrin are typically present in quantities of about 1×1011 greater than proteins and peptides of relatively low abundance. This massive difference in protein/peptide abundance as between high abundance and low abundance proteins/peptides can obfuscate the detection of small variations in proteins of low abundance. It is essentially a signal to noise ratio problem in the mass spectroscopy measurement, wherein the signatures of the lower concentration species are lost beneath the noise floor. The dynamic range of the analysis device (e.g., mass spectrometer) is at best 4-5, not close to the 11 orders of magnitude dynamic range of the serum proteome. In a typical mass spectrum based on a mammalian serum sample, there are number of proteins/peptides that are present in high abundance therein, as demonstrated by the series of large peaks in a mass spectra readout. The presence and quantity of the vast array of proteins/peptides of relatively low abundance are more difficult to discern.
This is especially problematic in conducting a comparative proteomic analysis of sera collected from multiple human subjects. The proteins and peptides of relatively high abundance in human sera are, generally speaking, the same for all human subjects (e.g., albumin, immunoglobulin and transferrin). It is frequently the presence and/or variation in quantity of proteins and peptides of relatively low abundance among different subjects that is most instructive in terms of gaining knowledge about human biology, medicine, disease and the like. Therefore, if the ability to discern the presence and/or variations in the quantity of proteins and peptides of low abundance is impeded, then the overall value of proteomic research in this field may ultimately be compromised.
There are a number of techniques commonly used in the art to “negatively select” the proteins and/or peptides of interest in a sample. These techniques may be used to help address the aforementioned problems. In negative selection techniques, unwanted molecules are removed from a sample, leaving behind only the proteins/peptides of interest. Examples of this type of selection include Enchant™ Life Science Kits for Albumin Depletion (available from Pall Corporation, New York, N.Y.), Agilent's Multiple Affinity Removal System via a Liquid Chromatography Column or as a Spin Cartridge (both available from Agilent Technologies, Palo Alto, Calif.), and high performance liquid chromatography.
However, negative selection techniques are oftentimes unsuccessful. Quantities of proteins and/or peptides of interest may be inadvertently removed from the sample along with the unwanted molecules when performing negative selection. Conversely, a significant volume of unwanted molecules frequently remains in the sample along with the proteins and/or peptides of interest. Either or both of these complications may lead to inaccurate or incomplete proteomic analyses of the resulting sample, and, in extreme situations, may render the samples entirely unusable.
There are positive selection methods based on glycosylation. For example, identification and quantification of peptides containing N-linked carbohydrates based on the conjugation of glycoproteins to a solid support medium using hydrazide chemistry, stable isotope labeling of the glycopeptides and specific release of formerly N-linked glycopeptides by peptide N-glycosidase F are available (Institute for Systems Biology; Seattle, Wash.). These methods reduce the dynamic range by several orders of magnitude but significant amounts of low abundance peptides are lost to analysis through this process. This technique is based on selection based on a post-translational modification, not on a quality of the actual peptide itself. There is therefore a need in the art for a technique to obtain a sample of proteins and/or peptides of interest for proteomic analysis that is substantially free from the aforementioned limitations of negative and crude positive selection procedures. There is a further need in the art for a procedure to ascertain a definitive blood chemistry signature for diagnostic and prognostic purposes; particularly if that procedure could alone serve as sufficient evidence for a wide range of medical diagnoses and prescriptions. Ideally, the procedure would return a blood chemistry signature that qualifies, to as great an extent as possible, the chemical composition of a patient's blood.